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Circ J. Author manuscript; available in PMC 2012 December 20.
Published in final edited form as:
Circ J. 2012 ; 76(1): 15–21.

Nitroglycerin Use in Myocardial Infarction Patients: Risks and

Benefits
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Julio C.B. Ferreira, PhD and Daria Mochly-Rosen, PhD

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford,
CA 94305-5174, USA

Abstract
Acute myocardial infarction and its sequelae are leading causes of morbidity and mortality
worldwide. Nitroglycerin remains a first-line treatment for angina pectoris and acute myocardial
infarction. Nitroglycerin achieves its benefit by giving rise to nitric oxide, which causes
vasodilation and increases blood flow to the myocardium. However, continuous delivery of
nitroglycerin results in tolerance, limiting the use of this drug. Nitroglycerin tolerance is due, at
least in part, to inactivation of aldehyde dehydrogenase 2 (ALDH2), an enzyme that converts
nitroglycerin to the vasodilator, nitric oxide. We have recently found that, in addition to
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nitroglycerin’s effect on the vasculature, sustained treatment with nitroglycerin negatively affects
cardiomyocyte viability following ischemia, thus resulting in increased infarct size in a myocardial
infarction model in animals. Co-administration of Alda-1, an activator of ALDH2, with
nitroglycerin improves metabolism of reactive aldehyde adducts and prevents the nitroglycerin-
induced increase in cardiac dysfunction following myocardial infarction. In this review, we
describe the molecular mechanisms associated with the benefits and risks of nitroglycerin
administration in myocardial infarction. (167 of 200).

Keywords
aldehyde dehydrogenase; nitric oxide; nitroglycerin tolerance; cardiomyocyte; cell death
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Benefits of nitroglycerin (glyceryl trinitrate or GTN)

Despite advances in pharmacological therapies, ischemic heart disease and acute myocardial
infarction (MI) continue to be a major cause of morbidity and death worldwide. According
to the World Health Organization, over 7 million people die of ischemic heart disease every
year. Consequently, novel pharmacological and non-pharmacological strategies need to be
explored to benefit MI patients.1–3

Since its discovery over 150 years ago, GTN has become the most common treatment for
patients with unstable angina pectoris, myocardial infarction and heart failure.4–8 The ability
of GTN to promote vasodilation as well as tolerance was clearly noted during the GTN
industry ascension in the 20th century. Factory workers, usually exposed to high levels of
organic nitrites, often complained of headaches on Mondays that disappeared over the
weekends. Indeed, factory workers suffering from angina pectoris or heart failure often

¶
Address correspondence to: Daria Mochly-Rosen, Ph.D., Department of Chemical and Systems Biology, Stanford University School
of Medicine, CCSR, Rm 3145A, 269 Campus Drive, Stanford, CA 94305-5174, Tel: 650-725-7720, Fax: 650-723-2253,
mochly@stanford.edu.
Conflict of interest: D.M.-R. is the founder of KAI Pharmaceuticals, Inc. However, none of the research in her laboratory is
supported by or is in collaboration with the company. J.C.B.F. has no disclosure.
 Ferreira and Mochly-Rosen Page 2

experienced relief from chest pain during the work week, but which recurred on weekends.
Both effects were attributed to the vasodilator action of GTN, which quickly became
apparent to physicians. The phenomenon of nitrate tolerance became famously recognized
by the onset of ‘Monday disease’ and of nitrate withdrawal/overcompensation by ‘Sunday
Heart Attacks’.9

The positive effects of GTN arise from its ability to promote vasodilation, resulting in
increased blood flow to the heart.10 GTN effects are also evident in systemic veins where
the venodilator effect reduces cardiac preload and further decreases myocardial wall
stress.11 GTN is extremely effective in restoring the equilibrium of oxygen and nutrients
supply-demand in the ischemic heart. However, sustained GTN administration causes
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tolerance and is associated with pro-oxidant effects, endothelial dysfunction and increased
sensitivity to vasoconstrictors.12–14 Other nitrates used in clinical practice include
isosorbide-dinitrate, its active metabolite isosorbide-mononitrate, pentaerythrytol
tetranitrate, erythrytyl-tetranitrate, and nicorandil.15

History of the therapeutic use of nitroglycerin

In 1847, working in Theophile-Jules Pelouze’s laboratory in Turin, Ascanio Sobrero
discovered GTN. Sobrero first noted the aggressive headache for several hours produced by
GTN.4 Two years later, knowing of Sobrero’s reports of headache, the German scientist
Constantin Hering tested GTN in healthy volunteers and observed that headache was caused
with much precision.4 Alfred Nobel joined Pelouze in 1851 and recognized the scientific
and financial potential of GTN. Years later, he began manufacturing GTN in Sweden. Nobel
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suffered from poor health for most of his life. In later life, he suffered from intense pain and
angina pectoris. It is therefore ironic that in 1890, his physicians recommended GTN for his
heart compliant.4

During the second half of the 19th century, several British scientists became interested in the
newly discovered amyl nitrite, recognized as a powerful vasodilator. Lauder Brunton used
the compound to relieve angina in 1867, and first reported the pharmacological resistance to
repeated doses.16, 17 Following Brunton’s work, scientists concentrated on recording the
effects of nitrite-containing compounds on several pathological systems, which include
angina pectoris, myocardial infarction, hypertension and heart failure.17–21 Finally, GTN
was established as a treatment for the relief of angina at the end of the 19th century.
However, the mechanism of action of GTN-induced benefit was discovered only 80 years
later.
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In the late 1970s, the vasodilator effect of GTN was discovered to be mediated by nitric
oxide, which was apparently generated from GTN in vascular smooth muscle.22–24 Years
later, it was discovered that mammalian cells synthesize nitric oxide.25 In 1998,
approximately 130 years after the invention of dynamite by Alfred Nobel and the first
observed clinical benefit of GTN, the Nobel Prize in Medicine or Physiology was awarded
for “Nitric Oxide as a Signaling Molecule in the Cardiovascular System” to Robert
Furchgott, Louis Ignarro and Ferid Murad. GTN remains the treatment of choice for
relieving angina; other organic esters and inorganic nitrates are also used, but the rapid
action of GTN and its established efficacy make it the mainstay of angina pectoris relief.4

ALDH2 in nitroglycerin bioactivation

The vasodilator action of GTN was discovered as a process mediated by nitric oxide.22, 24
Subsequent studies discovered that a chemical reaction between GTN (or other nitro
compounds) and a thiol generate an intermediate S-nitrosothiol, which resulted in further
production of nitric oxide.23 Nowadays, it is commonly assumed that GTN is converted in

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smooth muscle cells to nitric oxide which activates soluble guanylate cyclase to generate
cyclic GMP which, in turn, results in vascular smooth muscle relaxation (Figure 1).26

Despite intense basic and clinical research, the molecular mechanism by which NO is
generated from GTN remained elusive. In fact, it has been proposed that one or more
enzymatic reactions might be involved in GTN bioactivation.27–29 This research was
complicated because GTN bioactivation is mediated by either non-enzymatic or enzymatic
reactions. Endogenous reductants (i.e. thiols and ascorbate) can cause non-enzymatic GTN
bioactivation; however, their contribution to GTN-mediated vasodilation appears limited.30
Several enzymatic reactions are also involved in GTN bioactivation including glutathione-S-
transferases 31, xanthine oxidoreductase 32 and the cytochrome P450 system.26, 33, 34
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However, because none of these enzymes could catalyze the selective formation of 1,2-
glyceryl dinitrite (1,2-GDN) from GTN, the search for the GTN metabolizing enzyme
continued. In 2004, Stamler and collaborators identified mitochondrial ALDH2 as a key
enzyme catalyzing GTN bioconversion to 1,2- glyceryl dinitrite (1,2-GDN).26, 35

ALDH2 is a mitochondrial enzyme and a member of the NAD(P)+-dependent aldehyde

dehydrogenase family composed of 17 isozymes expressed with different tissue
distributions.36 ALDH2 is a tetrameric enzyme known mainly for its role in ethanol
metabolism, catalyzing aldehyde oxidation to form acetic acid.36–38 However, in addition to
its dehydrogenase activity, ALDH2 also has esterase and reductase activities, at least in
vitro.26 Based on the esterase activity of ALDH2, the reaction with GTN would be expected
to generate an S-nitrosothiol intermediate that would be hydrolyzed to produce nitrate.
However, nitrite formation indicates that S-nitrosothiol is reduced and not hydrolyzed.26
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Therefore, it has been suggested that thiols potentiate GTN activity through formation of
active S-nitrosothiol.39 In fact, purified ALDH2 catalyzes 1,2,-GDN formation only in the
presence of reducing agents.35

The role of ALDH2 as the GTN-reductase enzyme as well as its contribution to GTN-
induced vasodilation has been widely demonstrated over the last decade.13, 35, 40 Purified
mitochondrial ALDH2 catalyzes predominantly 1,2-GDN formation from low levels of
GTN (1 μM). In the presence of NAD+, however, the rate of GTN metabolism as well as the
1,2-GDN/1,3-GDN ratio drastically increases in either ALDH2 purified from bovine liver or
overexpressed human ALDH2.35, 41 Using different animal models, it has been shown that
inhibition of ALDH2 by chloral hydrate, disulfiram or cyanamide attenuates hypotension
induced by intravenous injection of GTN.35, 42 Treatment of aortic rings with ALDH2
inhibitors or substrates inhibits GTN-induced vasodilation.35, 41, 43, 44 Of interest, these
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inhibitors do not attenuate the sodium nitroprusside-induced relaxation (which directly

activates guanylate cyclase), suggesting that GTN-mediated effects are mainly through
specific inhibition of vascular ALDH2 activity.35 In ALDH2 knockout mice, the
hypotensive effect of GTN infusion was abrogated at low doses and substantially reduced at
high doses.45 As expected, GTN bioactivation to 1,2-GDN was mainly eliminated in
ALDH2 knockout mice. Together, all these studies support a critical role for ALDH2 in
GTN bioactivation.

ALDH2 contains three cysteine residues within the catalytic site (Cys 301, 302 and 303).
Stamler and collaborators proposed a specific mechanism for ALDH2 reductase activity
based on the formation of a disulfide between Cys 302 (the Cys that participates in the
catalysis) and one of the two adjacent Cys residues.35 Therefore, Mayer and collaborators
proposed that GTN denitration and bioactivation reflect two separate pathways of ALDH2-
catalyzed GTN biotransformation, both of which involve formation of a thionitrate
intermediate at Cys302 as initial reaction step.46 However the molecular mechanisms

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underlying this fractional contribution to ALDH2-catalyzed GTN bioconversion are still

unknown.

The hypothesis that nitrite produced by GTN-reductase activity of mitochondrial ALDH2 is

further reduced to generate nitric oxide, which activates soluble guanylate cyclase and
promotes vasodilation is well accepted (Figure 1). However, the molecular mechanisms of
nitrite conversion to nitric oxide remain controversial. Chen and collaborators demonstrated
that incubation of isolated mitochondria from fibroblasts with different concentrations of
GTN resulted in the dose-dependent generation of nitric oxide, where ALDH2 inhibition
blocked this response.45 It has been reported that mammalian mitochondria present a nitrite
reductase activity, associated with complex III and IV of the mitochondrial respiratory
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chain.47, 48 However, the mechanism for the three-electron reduction of GTN to generate
nitric oxide remains unknown. Recent studies demonstrated that mitochondria respiratory
chain is not involved in bioactivation of GTN-derived nitrite.41, 49

Mechanisms involved in nitroglycerin tolerance

GTN bioactivation in vascular smooth muscle is required to promote effective
vasorelaxation treatment of angina pectoris and congestive heart failure. However, the
usefulness of GTN is widely limited by the development of tolerance to the drug. Sustained
GTN therapy-induced tolerance is at least partially attributed to impaired bioactivation of
GTN.44 Moreover, GTN therapy causes a cross-tolerance, which results in impaired
relaxation by other nitrosovasodilators. Despite its multifactorial causes, including
desensitization of soluble guanylate cyclase, cGMP-dependent kinase and myosin light
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chain phosphorylation, GTN tolerance is mainly mediated by the inability of ALDH2 to

catalyze the conversion of GTN to 1,2-GDN and nitrite within mitochondria 26. For
example, chloral hydrate, a substrate analog ALDH2 inhibitor, and the specific ALDH2
inhibitor, daidzin, suppress 1,2-GDN formation by purified ALDH2.35, 41, 44 Also,
acetaldehyde competitively inhibits GTN turnover by ALDH2 both in vitro and in vivo.35, 44

The mechanism involved in ALDH2 inactivation may be a result of oxidation and formation
of a disulfide bond that includes the active site Cys thiol (Figure 1).26 As mentioned above,
the catalytic sites of ALDH2 contain three adjacent sulphydryl groups that can form
intramolecular disulfide bonds 50, which leads to inactivation of ALDH2.51 Active site
disulfide formation underlies the inhibition of ALDH2 by GTN, nitric oxide, its substrates
such as 4-hydroxynonenal as well as by other ALDH inhibitors.3550, 51 Analysis of purified
ALDH2 in vitro indicates that reduction of the oxidized enzyme by exogenous thiols or
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other reductants restores ALDH2 activity.35, 52

Mitochondrial ALDH2 also become a convergence point of the superoxide hypothesis of

GTN tolerance. Superoxide and peroxynitrite directly inhibit ALDH2 activity.44 Daiber et
al. (2005) observed that GTN-stimulated superoxide production correlated well with
decreases in ALDH2 activity.53 Moreover, reactive oxygen species production, mediated by
mitochondrial respiration blockade, was associated with mitochondrial ALDH2
inactivation.54 Sydow and collaborators reported that progression of GTN tolerance in an
animal model was directly associated with inhibition of vascular ALDH-2 activity,
disruption of GTN metabolism, and increased reactive oxygen species production by
mitochondria.44 Of interest, the increase in cGMP in response to GTN was blunted in
cultured endothelial cells deficient in mitochondria. These findings support the hypothesis
that the reactive oxygen species release and further accumulation of reactive aldehydes such
as 4-hydroxy-2-nonenal may contribute directly to GTN tolerance, either by oxidative
inhibition of ALDH2 55 or by oxidizing enzyme cofactors.56 In fact, superoxide-induced
inhibition of key enzymes related to bioactivation of other organic nitrates, or of

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downstream elements in the vasorelaxation signaling pathways, may contribute to GTN-

mediated cross-tolerance.26 Of interest, incubation of tolerant tissue with different reducing
agents or antioxidant molecules improved vascular ALDH2 activity, thus supporting the
hypothesis that enzyme oxidation leads to GTN tolerance.

It was recently shown that Alda-1 (a selective ALDH2 activator) may slightly inhibit GTN
bioconversion to NO, in vitro 57, which is not attractive considering the medical benefit of
NO in patients with angina pectoris. However, we found that Alda-1 given concomitantly
with GTN in vivo did not inhibit vasodilatation 52, indicating that Alda-1 does not inhibit
GTN bioactivation to NO in vivo. Moreover, we found that Alda-1 prevented ALDH2
inactivation due to prolonged treatment with GTN (Figure 2).
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Effects of GTN on cardiac cells

The effects of GTN in the vasculature have been widely investigated, but relatively little is
known about GTN’s effect on cardiac cells. We have recently demonstrated that sustained
treatment with GTN resulted in an increase in infarct size and cardiac dysfunction after
myocardial infarction in rats.52, 55 GTN tolerance-mediated deleterious effects in the heart
are associated with ALDH2 inactivation.55 As previously reported, GTN treatment
drastically inhibits the dehydrogenase activity of recombinant ALDH2, in vitro and in
vivo.52 We also found that co-incubation with GTN and Alda-1 (a selective ALDH2
activator that we have identified) completely prevented GTN-induced recombinant ALDH2
inactivation. Further, sustained treatment with GTN significantly reduced mitochondrial
ALDH2 activity in the rat myocardium, resulting in increased cardiac damage and
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ventricular dysfunction after a myocardial infarction event.52 Of interest, co-treatment with

GTN and Alda-1 restored ALDH2 activity, resulting in smaller infarct size and improved
cardiac function in rats.52 We suggest that GTN tolerance is the main process involved in
increased cardiac damage following MI, since the use of isosorbide dinitrate, an alternative
NO donor often used in a sustained fashion to treat angina, did not cause ALDH2
inactivation and further cardiac damage in vivo.52 Similarly, Sydow et al. observed that in
vivo treatment with GTN leads to reduced cardiac GTN biotransformation by mitochondrial
ALDH2 and resulted in accumulation of reactive oxygen species, where incubation of
mitochondria from tolerant animals with reducing agents restored ALDH2 function.44 These
findings suggest that patients under continuous GTN treatment are at risk for increased
cardiac damage.

Cellular side effects of GTN

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The deleterious effects of organic nitrate therapy on mitochondrial were first described 55
years ago, where acute GTN exposure was described to induce mitochondrial swelling, to
stimulated oxygen consumption and to caused loss of respiratory control of rat liver and
heart mitochondria.58 More recently, it was demonstrated that GTN infusion resulted in
mitochondrial dysfunction-induced oxidative stress in both animal and human blood
vessels.59–61 An excessive reactive oxygen species production along with reduction of more
than 50% in ALDH2 activity was observed in isolated mitochondria using the complex III
inhibitor antimycin A.62, 63 Mitochondrial-target antioxidants prevent complex I inhibition
mediated by GTN treatment.54 Therefore, accumulation of reactive aldehydes derived from
oxidative stress may disrupt GTN bioactivation by negatively targeting ALDH2 function.
Altogether, these findings suggest that GTN bioactivation requires functionally active
mitochondria, since increased reactive oxygen species production due to mitochondrial
dysfunction results in impaired ALDH2 activity and further GTN conversion.35, 44, 54

We have recently found that sustained GTN treatment significantly decreases aldehyde
dehydrogenase activity in the failing heart.52 The fact that mammalian ALDH2 functions as

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a GTN reductase may explain the inhibitory effect of GTN on the aldehyde dehydrogenase
activity. These findings point to a possible alcohol-GTN drug interaction through ALDH2
inactivation, which can result in a devastating phenomena induced by accumulation of
reactive aldehydes inside the cell. However, this hypothesis needs to be better explored.

ALDH2*2 mutation and nitroglycerin metabolism

Over 40% of East Asians carries a common ALDH2*2 mutation. The mutation is caused by
a single nucleotide substitution in exon 12 of the ALDH2 gene on chromosome 12, resulting
in an amino acid change from glutamate to lysine at position 487 (E487K) of the mature
enzyme.64 The E487K amino acid substitution at the interface of the tetrameric enzyme
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leads to a reduction of the catalytic ALDH2 activity due to a disruption of co-enzyme NAD
binding.65 Heterozygous ALDH2*1/*2 and homozygous ALDH*2/*2 show approximately
40% and 5% of wild-type ALDH2 activity, respectively.66 Epidemiological studies have
indicated that ALDH2 is crucial in cardiovascular diseases, where the ALDH*2/*2 allele has
been linked to higher incidences of GTN tolerance 40, myocardial infarction 67, 68 and
hypertension.69, 70

Considering the contribution of ALDH2 to the bioactivation of GTN, it is expected that the
significant decrease of ALDH2 activity in Asians carrying the E487K mutation would lead
to a decreased vasodilation response to GTN treatment. In fact, in vitro experiments
demonstrated that GTN biotransformation to 1,2-GDN was drastically decreased in the
E487K mutant enzyme.57 Subjects carrying the ALDH*2/*2 mutation have a significantly
reduced vasodilatory response to GTN, where larger doses of GTN were required to achieve
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satisfactory vasodilation.71 Of interest, treatment of non-ALDH2 mutant individuals with

the ALDH2 inhibitor, disulfiram, blocked the vasodilatory effect of GTN, but not that
promoted by sodium nitroprusside.71 These findings confirm that ALDH2 is crucial to GTN
bioactivation in humans and that individuals with the ALDH*2/*2 mutation display a
reduced vasodilatory response to GTN and are probably more prone to develop GTN
tolerance-associated cardiotoxicity.

Clinical Implications/summary
The findings that ALDH2 is crucial to GTN bioactivation may have important clinical
implications. Considering that ALDH2 activation positively correlates with cardioprotection
during ischemic events 55, therapies which reduce ALDH2 activity (i.e. sustained GTN
administration) may worsen outcome during an ischemic episode.
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GTN is among the most commonly used drugs in the treatment of angina, ischemia and
heart failure.4–8 However, there is discussion regarding the deleterious effect of chronic
GTN treatment.12, 36, 72 Given the importance of ALDH2 in cytoprotective signaling in
different tissues (including the heart), GTN tolerance should not be considered as a simple
loss of drug efficacy. The current clinical practice of GTN treatment in patients at elevated
risk for an ischemic event should be reviewed. Sustained exposure to GTN should be
avoided, not only because its benefit wears out, but perhaps more importantly, because it
decreases the activity of ALDH2 and thus exacerbates damage associated with ischemia.

A drug that can enhance GTN bioconversion and prevent the inhibitory effect of GTN on
ALDH2 will be very helpful, in particular for ALDH*2/*2 individuals. Activators of
ALDH2, such as Alda-1, may have therapeutic potential due to their ability to prevent GTN-
induced ALDH2 inactivation. The fact that Alda-1 restores activity of mutant ALDH*2/*2
suggests that these individuals may become more responsive to GTN treatment following
Alda-1 administration (Figure 3).

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Finally, further studies to clarify the molecular mechanisms of GTN biotransformation and
GTN tolerance, as well as clinical studies re-evaluating the use of sustained GTN treatment
in patients with cardiovascular diseases, especially in East Asian patients, are needed.

Acknowledgments
Funding

This study was supported by National Institute of Health Grant AA11147 and HL52141 to DMR. JCF holds a post-
doctoral fellowship from Fundação de Amparo a Pesquisa do Estado de São Paulo - Brasil (FAPESP
2009/03143-1).
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Figure 1.
Tolerance to GTN (nitroglycerin) is mainly caused by oxidative inactivation of the reductase
activity of ALDH2, which results in decreased GTN bioactivation, diminished levels of
nitrite (NO2-) and nitric oxide (NO). This response abrogates the GTN-induced vasodilation
phenomenon due to insufficient activation of soluble guanylate cyclase and production of
cyclic guanosine monophosphate (cGMP) in the vascular smooth muscle. Besides its
reductase activity, the dehydrogenase activity of ALDH2 is also down-regulated by GTN
tolerance, leading to accumulation of toxic aldehydes (i.e. 4-hydroxy-2-nonenal; 4-HNE)
inside the mitochondria, which further disrupts the mitochondrial respiratory chain and
results in reactive oxygen species generation. GTN-mediated ALDH2 inactivation triggers
apoptosis in cardiomyocytes as well as abolishes the vasodilator effect of GTN vascular
smooth muscle.
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Figure 2.
Alda-1 (a selective ALDH2 activator) increases dehydrogenase activity of ALDH2. The
contribution of Alda-1 to the reductase activity of ALDH2 has not yet been determined.
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Figure 3.
The ALDH2 mutation caused by a single amino acid change from glutamate to lysine at
position 487 (E487K) leads to a reduction in both the dehydrogenase and reductase activities
of ALDH2. Alda-1 significantly improves dehydrogenase activity of the mutant enzyme,
causing an allosteric change that corrects the structural defect in the enzyme.73 The effect of
Alda-1 on the reductase activity of ALDH2 has not yet been determined.
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